Timing of Epimerization and Condensation Reactions in Nonribosomal Peptide Assembly Lines: Kinetic Analysis of Phenylalanine Activating Elongation Modules of Tyrocidine Synthetase B

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Abstract
The cyclic decapeptide antibiotic tyrocidine has d-Phe residues at positions 1 and 4, produced during peptide chain growth from l-Phe residues by 50 kDa epimerase (E) domains embedded, respectively, in the initiation module (TycA) and the TycB3 module of the three-subunit (TycABC), 10-module nonribosomal peptide synthetase. While the initiation module clearly epimerizes the aminoacyl thioester Phe1-S-TycA intermediate, the timing of epimerization versus peptide bond condensation at internal E domains has been less well characterized in nonribosomal peptide synthetases. In this study, we use rapid quench techniques to evaluate a three-domain (ATE) and a four-domain version (CATE) of the TycB3 module and a six-domain fragment (ATCATE) of the TycB2-3 bimodule to measure the ability of the E domain in the TycB3 module to epimerize the aminoacyl thioester Phe-S-TycB3 and the dipeptidyl-S-enzyme (l-Phe-l-Phe-S-TycB3 ⇔ l-Phe-d-Phe-S-TycB3). The chiralities of the Phe-S-enzyme and Phe-Phe-S-enzyme species over time were determined by hydrolysis and chiral TLC separations, allowing for the clear conclusion that epimerization in the internal TycB3 module occurs preferentially on the peptidyl-S-enzyme rather than the aminoacyl-S-enzyme, by a factor of about 3000/1. In turn, this imposes constraints on the chiral selectivity of the condensation (C) domains immediately upstream and downstream of E domains. The stereoselectivity of the upstream C domain was shown to be l-selective at both donor and acceptor sites (LCL) by site-directed mutagenesis studies of an E domain active site residue and using the small-molecule surrogate d-Phe-Pro-l-Phe-N-acetylcysteamine thioester (d-Phe-Pro-l-Phe-SNAC) and d-Phe-Pro-d-Phe-SNAC as donor probes.